Experimental methods are being developed for the precise localization of small gaps in duplex DNA molecules. Our initial efforts will be to determine with high resolution the location of the gap in the viral strand of 0X174 RF during single strand synthesis. We plan to accomplish this by selective hydroxylamine mutagenesis of the DNA in the single stranded gap. Under appropriate conditions, the double stranded portion of the RF 11 will be unaffected by hydroxylamine. Mutants will be analyzed by both complementation and recombination. The gene 2 protein of M13 will be isolated and characterized by standard methods of enzymology. Purified gene 2 protein will be used in electron microscope heteroduplex experiments to determine the origin of M13 DNA replication.